Applications for electrophoresis, an analytical technique for separating and identifying constituents in a sample, include the determination of a sample's purity, the determination of molecular weights for proteins and nucleic acids, the mapping of nucleic acid primary structures, i.e. DNA and RNA sequence analyses, and the definition of phenotypic variance of a protein at the molecular level. Electrophoretic techniques rely on the fact that each molecular species has a unique combination of mass, size, shape, charge, density and sub-unit structure, all of which result in mobility differences responsive to an electric field. Various electrophoretic techniques use one or more of these properties to cause varying degrees of molecular separation via the migration of molecular species under an electric field.
Capillary electrophoresis is a technique using a capillary tube which is filled with a conductive fluid, as for example a buffer solution. A small amount of sample is introduced at one end of the capillary tube, whereafter a high potential difference is applied across the ends of the tube. Electroosmotic flow and differences in electrophoretic mobilities combine to provide a spatial separation of constituents of the sample solution at the outlet end of the capillary tube.
Electroosmotic flow is the movement of a liquid relative to a stationary charge surface as a result of electric fields applied to the liquid. U.S. Pat. No. 4,936,974 to Rose et al. explains electroosmotic flow as a result of charge accumulation at the interior capillary surface due to preferential adsorption of anions from the buffer solution that fills the bore of the capillary tube. The negative charge of the anions attracts a thin layer of mobile positively charged buffer ions which then accumulate adjacent to the inner surface. The charge accumulation at the interior wall provides a radially extending electric field. The potential across this radially extending electric field is referred to as the "zeta potential." The longitudinally extending electric field that is applied across the capillary tube attracts the positive ions which are hydrated by water toward a grounded outlet end of the capillary tube, viscously dragging other hydrated molecules. This dragging of molecules applies to neutral and negatively charged molecules, as well as positively charged molecules. The result is a bulk flow of the sample in the buffer solution toward the grounded outlet end of the capillary tube. Consequently, electroosmotic flow provides a means for moving neutral and negatively charged constituents of a sample toward a ground electrode.
Electrophoretic migration is the movement of charged constituents in response to an electric field applied along the longitudinal axis of the capillary tube. A positively charged molecule will be accelerated through the electroosmotic flow toward the ground electrode. Negatively charged molecules may be repelled by the ground electrode, but the force of the electroosmotic flow overcomes the repulsion and advances the negatively charged molecules.
As a result of the combination of electroosmotic flow and electrophoretic migration for an analysis in which a positive electrode is applied to the inlet end of the capillary tube and a ground electrode is applied to the outlet end, a spatial separation will occur with positively charged constituents exiting first, followed by neutral constituents and then negatively charged constituents. Each constituent of a sample may be identified by detecting the time required for the constituent to travel through the capillary tube. The quantity of the constituent within the sample is determined by the height and/or area of a signal trace on an electropherogram during a period of detection of that constituent.
An "on-column detector" detects migration of sample constituents past a detection area of the capillary tube between the inlet end and the outlet end. Ultraviolet and fluorescence on-column detectors are common. Alternatively, detection can take place after release of the sample from the outlet end, i.e., "off-column detection." For example, U.S. Pat. Nos. 4,705,616 to Andresen et al. and 4,842,701 to Smith et al. describe electrospraying the separated solution from the outlet end for off-column detection by mass spectrometry.
Obtaining an accurate analysis requires that each sample constituent be moved to the detection area. Often, the sample is introduced into the inlet end of the capillary tube by insertion of the inlet end into a sample vial, whereafter the inlet end is inserted into a first buffer vial electrically connected to a high voltage electrode. The outlet end of the capillary tube is inserted into a buffer reservoir vial connected to the ground electrode. Upon initiating the separation procedure, a negatively charged molecule may be drawn into the first buffer vial before electroosmotic flow can take full effect. Thus, these molecules will not be detected, rendering the analysis less accurate. Another problem in obtaining an accurate analysis involves the resolution of constituent detections. If a sample contains a number of constituents having similar electrophoretic mobilities, an analysis may be susceptible to errors in identifying and in quantifying the constituents. Yet another problem involves external factors, such as atmospheric conditions, that may have an effect on the electrophoretic separation.
U.S. patent application Ser. No. 07/754,797, to Young et al. which is assigned to the assignee of the present application and is incorporated herein by reference, describes a system and method for controlling electroosmotic flow and reducing undesired effects of external influences, thereby improving the analytic procedure. The rate of electroosmotic flow is directly proportional to the permittivity of the solution, the longitudinal axial electrical field and the zeta potential and is inversely proportional to the viscosity of the solution. Young et al. teach that the zeta potential can be controlled by providing a coating of electrically conductive material on the outside wall of the capillary tube. The conductive coating reduces the likelihood that an undesired voltage gradient will be created along the outside wall. A controlled field along the outside wall prevents external forces from affecting the internal ionic charge at the interior wall of the capillary tube. The electrically conductive coating may be allowed to float, but preferably is grounded to reduce the likelihood of electrostatic charges on the outside wall of the capillary tube.
An object of the present invention is to provide a capillary column of an electrophoresis system having on-column detection capability, wherein the capillary column provides improved control of electroosmotic flow.